A method for preparing aflatoxin lysine adduct based on solid-phase synthesis

Abstract

This invention belongs to the field of solid-phase synthesis technology. A method for preparing aflatoxin lysine adduct based on solid-phase synthesis includes the following steps: (1) synthesis of an amino acid-supporting resin; (2) deprotection of the amino acid resin to obtain N-α-Boc-lysine resin; (3) cyclization to obtain N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin; (4) oxidation to obtain N-ε-aflatoxin-N-α-Boc-lysine resin; (5) cleavage and purification to obtain the aflatoxin lysine adduct. This invention provides an aflatoxin lysine adduct. This invention also provides a standard for the aflatoxin lysine adduct. This invention utilizes solid-phase synthesis technology to synthesize aflatoxin amino acid adducts on resins, especially the efficient synthesis of AFB1-Lys, providing reliable standards for clinical medical diagnosis and food testing. Particularly in the determination of serum albumin aflatoxin adducts, it greatly assists in the accurate monitoring of aflatoxin exposure levels.

Description

Technical Field

[0001] This invention belongs to the field of solid-phase synthesis technology, specifically relating to a method for preparing aflatoxin lysine adducts based on solid-phase synthesis. Background Technology

[0002] Aflatoxins are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus. There are many types, with more than 12 identified so far. Common ones include aflatoxin B1, B2, G1, G2, and M1, among which aflatoxin B1 is the most widespread, most toxic, and most harmful. Although aflatoxin exposure can be predicted through dietary analysis, the determination of serum albumin aflatoxin adducts can accurately monitor aflatoxin exposure levels, which is of great significance in clinical medical diagnosis and food safety. Currently, a relatively effective method, such as DB34 / T 3861-2021, involves enzymatically digesting human serum albumin aflatoxin adducts in plasma with streptomycin, followed by solid-phase extraction of AFB1-Lys, and then using fluorescence detection. However, the complex synthesis process of AFB1-Lys, the lack of commercially available standards, and the inherent instability of AFB1-Lys itself pose significant challenges to the detection of aflatoxin exposure in blood samples.

[0003] Regarding the synthesis of AFB1-Lys, there are currently two main synthetic methods: the pre-oxidation method and the post-oxidation method. Several papers have reported on the pre-oxidation method. The basic process involves first oxidizing AFB1 to obtain the reactive epoxy intermediate aflatoxin B1-8,9-exo-epoxide (AFB1-8,9-exo-epoxide), which is then hydrolyzed to a diol and coupled with lysine to obtain AFB1-Lys. In 2022, Renaud et al. reported obtaining AFB1-Lys from AFB1 via the coupling of AFB1-dialdehyde and lysine under oxidative conditions such as hydrogen peroxide, by controlling the pH value.

[0004] When AFB1 binds to a protein, the amino group at the ε-position of lysine residues primarily participates in the reaction, while the amino group at the α-position maintains the peptide chain backbone. In existing reported synthetic methods, unprotected lysine residues are directly used as starting materials, resulting in two isomers of the product, making separation difficult and hindering the acquisition of high-quality standards.

[0005] Therefore, existing technologies urgently need to be addressed. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing aflatoxin lysine adducts based on solid-phase synthesis. This method avoids the use of highly carcinogenic aflatoxin B1-8,9 external epoxides in traditional synthetic routes, solves the reaction selectivity problem of aflatoxin B1-lysine adducts, achieves high purity and high yield of the product, reduces isomer formation, and yields high-quality standards. Furthermore, this method can be used to obtain lysine adducts of other toxins with similar structures.

[0007] The objective of this invention can be achieved through the following technical solutions: A method for preparing aflatoxin lysine adduct based on solid-phase synthesis includes the following steps: (1) Synthesis of amino acid carrier resin: The carrier resin was swollen with a good solvent to obtain a swollen resin; N-α-Boc-N-ε-Fmoc-lysine was weighed and added to an organic solvent and an organic base. After shaking to form a clear homogeneous solution, it was added to the swollen resin for reaction. The mixture was shaken, filtered, washed, and sealed with a blocking solution. The mixture was shaken until the reaction was complete, filtered, and washed to obtain N-α-Boc-N-ε-Fmoc-lysine resin. (2) Deprotection of amino acid resin: Using the N-α-Boc-N-ε-Fmoc-lysine resin prepared in step (1) as raw material, add piperidine organic solvent solution to treat 1 to 3 times to remove Fmoc protecting group, wash, and obtain N-α-Boc-lysine resin. (3) Cycloning: Weigh aflatoxin dialdehyde, add it to an organic solvent and an organic base, shake to form a clear homogeneous solution, and then add it to the N-α-Boc-lysine resin prepared in step (2). Shake, filter, and wash to obtain N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin. (4) Oxidation: Add organic solvent and oxidant to the N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin prepared in step (3), shake, filter, and wash to obtain N-ε-aflatoxin-N-α-Boc-lysine resin. (5) Cutting and purification: Using the N-ε-aflatoxin-N-α-Boc-lysine resin prepared in step (4) as raw material, acid lysis buffer is added to cut the aflatoxin lysine adduct on the N-ε-aflatoxin-N-α-Boc-lysine resin and remove the Boc protecting group at the same time. After filtration, vacuum rotary evaporation is performed, and the filtrate is added to the precipitant. After low-temperature centrifugation, the supernatant is discarded and the crude product is washed to obtain the crude product. The crude product is purified to obtain the aflatoxin lysine adduct.

[0008] In the preparation method of aflatoxin lysine adduct as described above, in step (1), the sample-carrying resin is selected from either CTC resin or Wang resin.

[0009] In the aflatoxin lysine adduct preparation method described above, the organic solvents in steps (1), (2), (3) and (4) are each independently selected from any one or a mixture of several of dichloromethane, dichloroethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone.

[0010] In the aflatoxin lysine adduct preparation method described above, in steps (1) and (3), each organic base is independently selected from any one or a mixture of several of N,N-diisopropylethylamine, triethylamine, triethanolamine, and pyridine.

[0011] In the aflatoxin lysine adduct preparation method described above, in step (5), the precipitant is icy ethyl ether or icy methyl tert-butyl ether.

[0012] In the preparation method of aflatoxin lysine adduct as described above, in step (5), the purification conditions are as follows: using a C18 reversed-phase column as the stationary phase, and using an aqueous solution containing 0.05% to 0.15% trifluoroacetic acid and an acetonitrile solution containing 0.05% to 0.15% trifluoroacetic acid as the mobile phase for gradient elution.

[0013] Based on the same inventive concept, the present invention provides an aflatoxin lysine adduct prepared by the preparation method described above, wherein the adduct is AFB1-Lys or AFG1-Lys, and its HPLC purity is not less than 95%.

[0014] Based on the same inventive concept, the present invention provides an aflatoxin lysine adduct standard, wherein the standard contains an aflatoxin lysine adduct prepared by the preparation method described above or an aflatoxin lysine adduct prepared by the method described above.

[0015] Based on the same inventive concept, this invention provides the application of aflatoxin lysine adduct prepared by the preparation method described above, or aflatoxin lysine adduct prepared by the preparation method described above, in the preparation of standards for the detection of aflatoxin exposure biomarkers.

[0016] Based on the same inventive concept, this invention provides the application of aflatoxin lysine adducts prepared by the preparation method described above, or aflatoxin lysine adducts prepared by the method described above, in the preparation of reagents or kits for food safety testing or clinical medical diagnosis.

[0017] Compared with existing technologies, the effects and advantages of this invention are: 1. This invention provides a method for preparing aflatoxin-lysine adducts based on solid-phase synthesis, which avoids the key intermediate of highly active and highly carcinogenic aflatoxin B1-8,9-external epoxide that must be used in existing pre-oxidation methods. It provides a safe and rapid synthesis scheme and preparation method for aflatoxin-lysine standards, successfully bypassing the highly toxic, unstable and extremely risky intermediate step, making the process easier to carry out safely under conventional laboratory conditions.

[0018] 2. This invention provides a method for preparing aflatoxin lysine adducts based on solid-phase synthesis. Addressing the long-standing selectivity problem of generating difficult-to-separate mixtures in existing technologies, this invention introduces a clever amino protection strategy to achieve precise control of the reaction site, ensuring that the reaction occurs only at the target ε-amino group, thereby completely eliminating the formation of non-target isomers at the source. This design solves the core pain points of existing technologies, such as difficult product purification and difficulty in guaranteeing the purity of standards, and achieves high product purity and high yield, reducing isomer formation and lowering the difficulty of subsequent purification.

[0019] 3. The present invention provides a method for preparing aflatoxin lysine adducts based on solid-phase synthesis, which has good universality and scalability. The technical principle is not limited to the synthesis of AFB1-Lys, but also provides ideas and methods for the preparation of adducts of other fungal toxins (AFG1, etc.) with lysine or lysine-containing peptides with similar reaction sites. Attached Figure Description

[0020] Figure 1 The HNMR spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of this invention; Figure 2 The liquid phase spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of this invention; Figure 3 The mass spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of this invention; Figure 4 The liquid phase spectrum of aflatoxin G1-lysine prepared in step (5) of Example 3 of this invention; Figure 5 The mass spectrum of aflatoxin G1-lysine prepared in step (5) of Example 3 of this invention; Figure 6 The HNMR spectrum of the control isomer aflatoxin B1-lysine. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0023] A method for preparing aflatoxin lysine adduct based on solid-phase synthesis includes the following steps: (1) Synthesis of amino acid carrier resin: The carrier resin was swollen with a good solvent to obtain swollen resin; N-α-Boc-N-ε-Fmoc-lysine was weighed and added to an organic solvent and an organic base. After shaking to form a clear homogeneous solution, it was added to the swollen resin for reaction. The mixture was shaken, filtered, washed, and sealed with a blocking solution. The mixture was shaken, filtered, and washed to obtain N-α-Boc-N-ε-Fmoc-lysine resin. (2) Deprotection of amino acid resin: Using the N-α-Boc-N-ε-Fmoc-lysine resin prepared in step (1) as raw material, add piperidine organic solvent solution to treat 1 to 3 times to remove Fmoc protecting group, wash, and obtain N-α-Boc-lysine resin. (3) Cycloning: Weigh aflatoxin dialdehyde, add it to an organic solvent and an organic base, shake to form a clear homogeneous solution, and then add it to the N-α-Boc-lysine resin prepared in step (2). Shake, filter, and wash to obtain N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin; In this invention, aflatoxin dialdehyde is selected from at least one of aflatoxin B2a and aflatoxin G2a; (4) Oxidation: Add organic solvent and oxidant to the N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin prepared in step (3), shake, filter, and wash to obtain N-ε-aflatoxin-N-α-Boc-lysine resin. (5) Cutting and purification: Using the N-ε-aflatoxin-N-α-Boc-lysine resin prepared in step (4) as raw material, acid lysis buffer is added to cut the aflatoxin lysine adduct on the N-ε-aflatoxin-N-α-Boc-lysine resin and remove the Boc protecting group at the same time. After filtration, vacuum rotary evaporation is performed, and the filtrate is added to the precipitant. After low-temperature centrifugation, the supernatant is discarded and the crude product is washed to obtain the crude product. The crude product is purified to obtain the aflatoxin lysine adduct.

[0024] The chemical structural formulas of the aflatoxin lysine adduct are shown in formulas (I) and (II):

[0025] (Ⅰ)AFB1-LYS (Ⅱ)AFG1-LYS In some embodiments of the present invention, the reaction equation for a method for preparing aflatoxin lysine adduct based on solid-phase synthesis is as follows: (1) Synthetic route of AFB1-LYS:

[0026]

[0027] (2) Synthetic route of AFG1-LYS:

[0028]

[0029] Preferably, in step (1), the sample-carrying resin is selected from either CTC resin or Wang resin.

[0030] Preferably, in step (1), the sample-carrying resin is selected from CTC resin, wherein the degree of crosslinking of the CTC resin is 1% and the degree of substitution is 1.245 mmol / g. Under these conditions, CTC resin is selected because it is extremely sensitive to acid and can achieve rapid and quantitative cleavage under very mild acidic conditions (such as low concentration of trifluoroacetic acid), achieving mild and controllable final cleavage, minimizing damage to acid-sensitive amino acids, and improving the purity and yield of the target product. At the same time, the chlorine atom has extremely high reactivity, and when it undergoes esterification reaction with the carboxylate of the first amino acid, it can achieve near-quantitative loading efficiency, and can accurately control and obtain high loading through loading conditions over a wide range. In addition, its triphenylmethyl steric hindrance effect produces a significant "steric protection" effect, which can effectively suppress racemization side reactions that may occur during loading and subsequent coupling.

[0031] Preferably, the organic solvents in steps (1), (2), (3), and (4) are each independently selected from any one or a mixture of several of dichloromethane, dichloroethane, trichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone. Preferably, in step (1), the organic solvent is selected from dichloromethane. The low polarity and aprotic physicochemical properties of dichloromethane are highly compatible with the polystyrene resin matrix, and it can fully penetrate and weaken the hydrophobic cross-linked network structure inside the resin according to the principle of "like dissolves like," thereby enabling the resin to obtain an excellent and uniform swelling effect. The reaction sites on the resin are fully exposed, and it is ensured that the subsequent reaction reagents can be rapidly and uniformly diffused to each active site through unobstructed solvation microporous channels, thus fundamentally ensuring the high efficiency and uniformity of the coupling reaction and providing ideal conditions for the smooth loading of amino acids.

[0032] Preferably, in steps (2), (3), and (4), the organic solvent is selected from N,N-dimethylformamide. N,N-dimethylformamide is a polar proton inert solvent that does not provide acid protons, will not quench activation intermediates, and will not form highly active O-acylisoureas, active esters, or other byproducts with amino acids during activation, thus ensuring activation efficiency. Furthermore, N,N-dimethylformamide has a high boiling point and its volatility at room temperature is far lower than that of dichloromethane, reducing solvent evaporation loss and operator exposure risks. In addition, the organic solvent in step (4) is selected from N,N-dimethylformamide, ensuring that both the raw material and the oxidant are fully dissolved to form a homogeneous solution, resulting in a fast reaction rate.

[0033] Preferably, in steps (1) and (3), the organic base is independently selected from any one or a mixture of several of N,N-diisopropylethylamine, triethylamine, triethanolamine, and pyridine. Preferably, in steps (1) and (3), the organic base is selected from N,N-diisopropylethylamine. Because N,N-diisopropylethylamine can efficiently and rapidly catalyze the β-elimination reaction of the Fmoc protecting group in polar aprotic solvents such as N,N-dimethylformamide, ensuring that the deprotection step of each condensation cycle is completely completed, and at the same time, its strong basicity and moderate nucleophilicity achieve the best balance, which can maintain the basic environment required by the reaction system in the subsequent amino acid activation coupling step, while minimizing the racemization side reaction caused by the excessive nucleophilicity of the base.

[0034] Preferably, in step (1), the good solvent is selected from dichloromethane.

[0035] Preferably, in step (1), the swelling reaction time is 0.5 to 2 hours.

[0036] Preferably, in step (1), the molar ratio of the sample-carrying resin to N-α-Boc-N-ε-Fmoc-lysine is 1:(1-3).

[0037] Preferably, in step (1), the sealing solution is a mixed solvent of dichloromethane, methanol, and N,N-diisopropylethylamine. Preferably, the volume ratio of dichloromethane, methanol, and N,N-diisopropylethylamine is 18:2:1. Under the catalysis of the organic base N,N-diisopropylethylamine, methanol can undergo a highly efficient carbamate esterification reaction with the free amino group at the resin terminal, forming a stable methoxycarbonyl (-NH-COOCH3) protection. This reaction is mild, and the byproduct is volatile methanol, which can be removed by subsequent washing, avoiding the over-acylation or side reactions that may occur with the traditional anhydride method. N,N-diisopropylethylamine, as a non-nucleophilic strong base, efficiently catalyzes the addition reaction of methanol and amino groups, ensuring that the reaction system maintains the necessary alkaline environment, promoting complete reaction. At the same time, its sterically hindered structure prevents it from participating in unnecessary side reactions as a nucleophile.

[0038] Preferably, in step (1), the washing solvent is dichloromethane. Under these conditions, residual solvent and unloaded amino acids in the resin are washed by vacuum filtration to avoid affecting the purity of subsequent reactions and reduce impurity formation.

[0039] Preferably, in steps (1) and (3), the reaction time is 2 to 6 hours each.

[0040] Preferably, in step (2), the organic solvent solution of piperidine is an N,N-dimethylformamide solution of piperidine. Preferably, the N,N-dimethylformamide solution of piperidine is a solution with a volume ratio of piperidine to N,N-dimethylformamide of 2:8, that is, the N,N-dimethylformamide solution of piperidine is a 20% N,N-dimethylformamide solution by volume.

[0041] Preferably, in step (2), the washing solvent is N,N-dimethylformamide. Under this condition, the resin is washed 3 to 6 times with the washing solution after each removal of the protecting group to reduce piperidine solvent residue.

[0042] Preferably, in step (3), the molar ratio of the N-α-Boc-lysine resin to aflatoxin dialdehyde is 1:(1-2).

[0043] Preferably, in step (4), the oxidant is selected from any one or a mixture of several of peracetic acid, methyldioxyethylene, peroxytert-butanol, and m-chloroperoxybenzoic acid. Preferably, in step (4), the oxidant is selected from any one of peracetic acid and m-chloroperoxybenzoic acid. Most preferably, in step (4), the oxidant is selected from m-chloroperoxybenzoic acid, which is sensitive to impact and poses a risk of rapid combustion and explosion; therefore, it is added dropwise to the reaction solution in small batches during the reaction.

[0044] Preferably, in step (4), the mass-to-volume ratio of the N-ε-aflatoxin-N-α-Boc-lysine resin to the solvent is 1 g: (10-20 mL).

[0045] Preferably, in step (4), the ratio of the N-ε-aflatoxin-N-α-Boc-lysine resin to the oxidant is 1:(1-3).

[0046] Preferably, in step (4), the reaction conditions are a temperature of -10℃ to 15℃. Preferably, in step (4), the reaction conditions are a temperature of 0℃.

[0047] Preferably, in step (4), the reaction time is 0.5 to 2 hours. Preferably, in step (4), the reaction time is 1 hour, and the reaction time is strictly controlled within 2 hours. Too long a reaction time will lead to an increase in peroxidation byproducts.

[0048] Preferably, in step (5), the lysis buffer is any one of TFA / DCM, TFA / TIS / water, or TFA / phenol / water / TIS. Preferably, the lysis buffer is any one of the following: TFA / DCM with a volume ratio of 95:5, TFA / DCM with a volume ratio of 3:7, TFA / TIS / water with a volume ratio of 95:2.5:2.5, or TFA / phenol / water / TIS with a volume ratio of 88:5:5:2.

[0049] Preferably, in step (5), the volume ratio of the N-ε-aflatoxin-N-α-Boc-lysine resin to the lysis buffer is 1 g: (5-20 mL).

[0050] Preferably, in step (5), the reaction time is 10 to 60 minutes. Preferably, in step (5), the reaction time is 30 minutes to ensure that the amino acids are completely removed from the resin and that the Boc protecting group is also removed in the acid lysis solution.

[0051] Preferably, in step (5), the vacuum degree of the rotary evaporation is 0.08-0.09 MPa, the rotation speed is 30-80 rpm, and the temperature is 25-35℃; the filtrate is rotary evaporated to 1 / 4 to 1 / 2 of the residual original solvent. More preferably, the filtrate is rotary evaporated to 1 / 4 of the residual original solvent.

[0052] Preferably, in step (5), the washing solvent is dichloromethane, and the washing times are generally 2 to 7 times to ensure that there are no product residues on the resin that would affect the yield of subsequent reactions.

[0053] Preferably, in step (5), the precipitant is icy diethyl ether or icy methyl tert-butyl ether. Preferably, in step (5), the precipitant is icy diethyl ether, which usually refers to anhydrous diethyl ether pre-cooled at around 0°C. Most synthetic peptides have extremely low solubility in cold diethyl ether, thus achieving high-yield precipitation and reducing product loss. Icy diethyl ether can selectively dissolve and remove impurities such as cleavage reagents, protecting group fragments, and small organic molecule byproducts. Moreover, the high volatility of diethyl ether facilitates rapid removal through subsequent vacuum drying, thereby obtaining a crude peptide solid that is easy to further purify.

[0054] Preferably, in step (5), the precipitation condition is to allow the sediment to stand and cool at -20°C.

[0055] Preferably, in step (5), the CTC resin is selected from TFA / DCM with a volume fraction of 30% as the pyrolysis solution, and the Boc protecting group is removed at the same time as the pyrolysis resin; the Wang resin is selected from TFA / DCM with a volume fraction of 95% as the pyrolysis solution.

[0056] Preferably, in step (5), the purification conditions are as follows: gradient elution is performed using a C18 reversed-phase column as the stationary phase, mobile phase A being ultrapure water (containing 0.1% trifluoroacetic acid by volume) and mobile phase B being preparative grade acetonitrile (containing 0.1% trifluoroacetic acid by volume).

[0057] More preferably, in step (5), the gradient elution includes the following steps: setting the flow rate of the preparative liquid chromatograph to 25 mL / min, the initial mobile phase ratio to 5% mobile phase B, setting the elution time to 40 min, and the gradient to 5%–30% mobile phase B (0–25 min), 30%–50% mobile phase B (25–35 min), and 50%–5% mobile phase B (35–40 min).

[0058] In steps (1) to (5) of this invention, the reactor is a constant-temperature oscillator. A circulating water vacuum pump is used as a pressure reducing filter, with a pressure of 0.08 to 0.1 MPa.

[0059] This invention utilizes solid-phase synthesis technology to synthesize aflatoxin amino acid adducts on resins, especially the highly efficient synthesis of AFB1-Lys, providing reliable standards for clinical medical diagnosis and food testing. In particular, it greatly assists in the accurate monitoring of aflatoxin exposure levels in the determination of serum albumin aflatoxin adducts.

[0060] Based on the same inventive concept, this invention provides an aflatoxin lysine adduct prepared by the method described above, wherein the adduct is AFB1-Lys or AFG1-Lys, and its HPLC purity is not less than 95%. By replacing the substrate with other substrates in the aflatoxin family, corresponding lysine adducts can be synthesized.

[0061] Based on the same inventive concept, the present invention provides an aflatoxin lysine adduct standard, wherein the standard contains an aflatoxin lysine adduct prepared by the preparation method described above or an aflatoxin lysine adduct prepared by the method described above.

[0062] Based on the same inventive concept, this invention provides the application of aflatoxin lysine adduct prepared by the preparation method described above, or aflatoxin lysine adduct prepared by the preparation method described above, in the preparation of standards for the detection of aflatoxin exposure biomarkers.

[0063] Based on the same inventive concept, this invention provides the application of aflatoxin lysine adducts prepared by the preparation method described above, or aflatoxin lysine adducts prepared by the method described above, in the preparation of reagents or kits for food safety testing or clinical medical diagnosis.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0066] CTC resin, with a crosslinking degree of 1% and a substitution degree of 1.245 mmol / g, was purchased from Shanghai Dibai Biotechnology Co., Ltd. Wang resin, with a crosslinking degree of 1% and a substitution degree of 1.125 mmol / g, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. N-α-Boc-N-ε-Fmoc-lysine was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. The standard aflatoxin B2a (abbreviated as "AFB2a") is a standard product of Qingdao Purybang Biotechnology Co., Ltd.; The standard aflatoxin G2a (abbreviated as "AFG2a") is a standard product of Qingdao Pribang Biotechnology Co., Ltd.

[0067] Example 1 This embodiment provides a method for preparing aflatoxin lysine adduct based on solid-phase synthesis, including the following steps: (1) Synthesis of N-α-Boc-N-ε-Fmoc-lysine resin:

[0068] Weigh 1.68 g of CTC resin (1% crosslinking, 1.245 mmol / g degree of substitution) and add it to a 100 mL solid-phase synthesis tube. Add 30 mL of dichloromethane. Transfer the reaction tube containing the above solution to a constant-temperature shaker at 25 °C and shake evenly for 1 hour. It can be seen that the CTC resin swells in the solvent and the volume of the colloidal solution increases, indicating that the resin swelling is complete. Filter the solvent under reduced pressure and wash three times with 30 mL of dichloromethane. Next, N-α-Boc-N-ε-Fmoc-lysine (1 g, 2.13 mmol) was weighed and placed in a centrifuge tube. 30 mL of dichloromethane and N,N-diisopropylethylamine (549 mg, 4.26 mmol) were added. After shaking to form a clear homogeneous solution, the solution was added to the swollen resin. The solution was then transferred to a constant temperature shaker and shaken uniformly at 170 rpm and 25°C for 4 hours. After the reaction was completed, a trace amount of resin was used as a lysis buffer (100 μL of a mixture of trifluoroacetic acid and dichloromethane, volume ratio of trifluoroacetic acid to dichloromethane 5:95) to cleave the resin. The reaction was monitored by TLC and HPLC. After the reaction, the solvent was filtered under reduced pressure, and the resin was washed five times with 30 mL of dichloromethane. Then, 42 mL of a mixture of dichloromethane, methanol, and N,N-diisopropylethylamine (volume ratio of dichloromethane, methanol, and N,N-diisopropylethylamine 18:2:1) was added as a resin blocking solution to block the remaining unreacted active sites. The resin was then transferred to a constant-temperature shaker and shaken uniformly for 1 hour. After the reaction, the solvent was filtered under reduced pressure, and the resin was washed three times with 30 mL of dichloromethane and three times with 30 mL of N,N-dimethylformamide to obtain N-α-Boc-N-ε-Fmoc-lysine resin. The intermediate did not require purification and was directly added to the next step.

[0069] (2) Synthesis of N-α-Boc lysine resin:

[0070] In solid-phase synthesis, the molar ratio of feed in subsequent steps is calculated based on the degree of substitution of the resin in step (1) (i.e., the number of moles of functional groups on a unit mass of resin).

[0071] Using N-α-Boc-N-ε-Fmoc-lysine resin (1.245 mmol / g) as the raw material, 30 mL of a 20% piperidine N,N-dimethylformamide solution was measured and placed in a solid-phase synthesis tube. The solution was transferred to a constant-temperature shaker at 25°C and shaken at 170 rpm for 10 minutes. After the reaction, the solvent was filtered under reduced pressure, and the solution was washed five times with 15 mL of N,N-dimethylformamide. Another 30 mL of a 20% piperidine N,N-dimethylformamide solution was measured and shaken at 170 rpm for 10 minutes. After the reaction, the solvent was filtered under reduced pressure, and the solution was washed five times with 15 mL of N,N-dimethylformamide. Finally, the above steps were repeated three times to complete the deprotection and thorough washing with a 20% piperidine N,N-dimethylformamide solution. Dip a dropper into a test tube containing 10 mg of resin, add 300 μL of N,N-dimethylformamide, and then add 100 μL of ninhydrin detection solution. Heat at 100 °C for 1-2 min. If the solution turns deep blue, it indicates the presence of free amino groups, thus obtaining N-α-Boc lysine resin. The intermediate does not require purification and can be directly added to the next step.

[0072] (3) Synthesis of N-ε-AFB2a-N-α-Boc-lysine resin:

[0073] Using N-α-Boc-lysine resin (1.245 mmol / g) as the starting material, standard AFB2a (0.7 g, 2.13 mmol) was weighed and placed in a centrifuge tube. 30 mL of N,N-dimethylformamide and N,N-diisopropylethylamine (549 mg, 4.26 mmol) were added, and the mixture was shaken to form a clear homogeneous solution. This solution was then added to a solid-phase synthesis tube and transferred to a constant-temperature shaker at 25°C and 170 rpm for 4 hours. After the reaction, a trace amount of resin was used as a lysis buffer (100 μL of a mixture of trifluoroacetic acid and dichloromethane, volume ratio 5:95) for resin lysis, and the lysis was monitored by HPLC. After no residual starting material peak was observed, the solvent was filtered under reduced pressure, and the resin was washed five times with 15 mL of N,N-dimethylformamide to obtain N-ε-AFB2a-N-α-Boc-lysine resin. The intermediate did not require purification and was directly added to the next step.

[0074] (4) Synthesis of N-ε-AFB1-N-α-Boc-lysine resin:

[0075] Using N-ε-AFB2a-N-α-Boc-lysine resin (1.245 mmol / g) as the starting material, 30 mL of N,N-dimethylformamide was added to a solid-phase synthesis tube. The oxidant, m-chloroperoxybenzoic acid (0.74 g, 4.26 mmol), was weighed and placed in a centrifuge tube. 3 mL of N,N-dimethylformamide was added, and the mixture was slowly added dropwise to the solid-phase synthesis tube at 0°C. After the addition was complete, the tube was transferred to a constant-temperature shaker and shaken at 170 rpm for 1 hour at 0°C. After the reaction, a trace amount of resin was used to cleave the resin with 100 μL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio 5:95). HPLC monitoring showed no residual starting material peak. The solvent was then filtered under reduced pressure, and the resin was washed five times with 15 mL of N,N-dimethylformamide to obtain N-ε-AFB1-N-α-Boc-lysine resin. The intermediate did not require purification and was directly added to the next step.

[0076] (5) Synthesis of aflatoxin lysine adduct AFB1-Lys:

[0077] Using N-ε-AFB1-N-α-Boc-lysine resin (1.245 mmol / g) as the starting material, 20 mL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio of trifluoroacetic acid to dichloromethane 3:7) was added to a solid-phase synthesis tube as the lysis buffer. This step used a higher concentration of lysis buffer to remove the N-α-Boc protecting group while cleaving the resin. The reaction was transferred to a constant-temperature shaker and shaken at 25 °C and 170 rpm for 30 minutes. After the reaction, the lysis buffer was monitored by HPLC. If no residual starting material peak was observed, the filtrate was collected by vacuum filtration, washed five times with 10 mL of dichloromethane, and the filtrate was collected. Most of the solvent was removed by rotary evaporation at a vacuum of 0.08–0.09 MPa, a rotation speed of 40 rpm, and 25 °C. The filtrate was then evaporated until 1 / 4 of the original solvent remained. This filtrate was then added dropwise to 50 mL of ice-cold diethyl ether and cooled at -20 °C. A large amount of white precipitate was observed in the reaction solution. The precipitate was transferred to a 50 mL centrifuge tube and centrifuged at 5 °C, 10,000 rpm for 5 min. The supernatant was discarded, and the filter cake was washed 2-3 times with ice-cold ether. The resulting solid was the crude peptide product. The sample to be purified was dissolved in acetonitrile / water (1:1, v / v) at a concentration of 15 mg / mL, ensuring complete dissolution. The sample solution was filtered through a 0.22 μm organic phase filter membrane to remove particulate impurities and prevent column clogging. A C18 reversed-phase column (5 μm, 30 × 150 mm) was selected. Mobile phase A was ultrapure water (containing 0.1% trifluoroacetic acid by volume), and mobile phase B was preparative grade acetonitrile (containing 0.1% trifluoroacetic acid by volume). The mobile phases were degassed by sonication for 15 min and filtered through a 0.22 μm filter membrane. The preparative HPLC system was set to a flow rate of 25 mL / min, with an initial mobile phase composition of 5% mobile phase B. The elution time was set to 40 min, with a gradient of 5%–30% mobile phase B (0–25 min), 30%–50% mobile phase B (25–35 min), and 50%–5% mobile phase B (35–40 min). The product was collected using a fraction collector, lyophilized, and its HPLC purity was determined to be 99%, yielding a high-purity aflatoxin lysine adduct AFB1-Lys. The product was a pale yellow solid, yielding 805 mg, with an overall yield of 83%.

[0078] 1H NMR (500 MHz, D2O) δ 7.42 (s, 1H), 6.19 (s, 1H), 4.27 (s, 2H), 3.78(s, 3H), 3.73 – 3.67 (m, 1H), 3.53 (t, J = 7.3 Hz, 2H), 3.11 – 3.05 (m, 2H),2.46 (s, 2H), 1.99 – 1.89 (m, 2H), 1.72 (q, J = 7.6 Hz, 2H), 1.49 – 1.41 (m,2H).LC-MS m / z found for C 23 H 24 O8N2[M + H] + :457.22; [M + Na] + : 459.24. Example 2 The difference between this embodiment and the synthesis method in Example 1 is that the resin used in the reaction is Wang resin, which is relatively stable with amino acids under strong acid conditions, making it easier to try the peracetic acid oxidation conditions in solid-phase synthesis.

[0079] This embodiment provides a method for preparing aflatoxin lysine adduct based on solid-phase synthesis, including the following steps: (1) Synthesis of N-α-Boc-N-ε-Fmoc-lysine resin:

[0080] Weigh 1.68 g of Wang resin (1% crosslinking, 1.125 mmol / g degree of substitution), using the degree of substitution as a control for subsequent molar feed ratios, and add it to a 100 mL solid-phase synthesis tube. Add 30 mL of dichloromethane. Transfer the reaction tube containing the above solution to a constant-temperature shaker at 25°C and shake evenly for 1 hour. The Wang resin will swell in the solvent, and the volume of the colloidal solution will increase, indicating that the resin swelling is complete. Filter the solvent under reduced pressure and wash three times with 30 mL of dichloromethane. Next, 1 g (2.13 mmol) of N-α-Boc-N-ε-Fmoc-lysine was weighed and placed in a centrifuge tube. 30 mL of dichloromethane and N,N-diisopropylethylamine (549 mg, 4.26 mmol) were added. After shaking to form a clear homogeneous solution, the solution was added to the swollen resin. The solution was then transferred to a constant temperature shaker and shaken uniformly at 170 rpm and 25 °C for 4 hours. After the reaction was completed, 10 mg of resin was used as a lysis buffer with 100 μL of a mixture of trifluoroacetic acid and dichloromethane (the volume ratio of trifluoroacetic acid to dichloromethane was 95:5). The reaction was monitored by TLC and HPLC. After the reaction, the solvent was filtered under reduced pressure, and the resin was washed five times with 30 mL of dichloromethane. Then, 42 mL of a mixture of dichloromethane, methanol, and N,N-diisopropylethylamine (volume ratio of dichloromethane, methanol, and N,N-diisopropylethylamine 18:2:1) was added as a resin blocking solution to block the remaining unreacted active sites. The resin was then transferred to a constant-temperature shaker and shaken uniformly for 1 hour. After the reaction, the solvent was filtered under reduced pressure, and the resin was washed three times with 30 mL of dichloromethane and three times with 30 mL of N,N-dimethylformamide to obtain N-α-Boc-N-ε-Fmoc-lysine resin. The intermediate did not require purification and was directly fed into the next step.

[0081] (2) Synthesis of N-α-Boc lysine resin:

[0082] In solid-phase synthesis, the molar ratio of feed in subsequent steps is calculated based on the degree of substitution of the resin in step (1) (i.e., the number of moles of functional groups on a unit mass of resin).

[0083] Using N-α-Boc-N-ε-Fmoc-lysine resin (1.125 mmol / g) as the raw material, 30 mL of a 20% piperidine N,N-dimethylformamide solution was measured and placed in a solid-phase synthesis tube. The tube was transferred to a constant-temperature shaker at 25°C and the reaction was carried out at 170 rpm for 10 minutes. After the reaction, the solvent was filtered under reduced pressure, and the mixture was washed five times with 15 mL of N,N-dimethylformamide. Another 30 mL of a 20% piperidine N,N-dimethylformamide solution was measured and the mixture was shaken at 170 rpm for 10 minutes. After the reaction, the solvent was filtered under reduced pressure, and the mixture was washed five times with 15 mL of N,N-dimethylformamide. Finally, the above steps were repeated three times to complete the deprotection process with a 20% piperidine N,N-dimethylformamide solution and thorough washing. Dip a dropper into a test tube containing 10 mg of resin, add 300 μL of N,N-dimethylformamide, and then add 100 μL of ninhydrin detection solution. Heat at 100°C for 1-2 min. The solution turns deep blue, indicating the presence of free amino groups, thus yielding N-α-Boc lysine resin. The intermediate does not require purification and can be directly fed into the next step.

[0084] (3) Synthesis of N-ε-AFB2a-N-α-Boc-lysine resin:

[0085] Using N-α-Boc lysine resin (1.125 mmol / g) as the starting material, standard AFB2a (0.7 g, 2.13 mmol) was weighed and placed in a centrifuge tube. 30 mL of N,N-dimethylformamide and N,N-diisopropylethylamine (549 mg, 4.26 mmol) were added, and the mixture was shaken to form a clear homogeneous solution. This solution was then transferred to a solid-phase synthesis tube and transferred to a constant-temperature shaker at 25°C and 170 rpm for 4 hours. After the reaction, a trace amount of resin was cleaved using 100 μL of a mixture of trifluoroacetic acid and dichloromethane (95:5 volume ratio) as lysis buffer. HPLC monitoring was performed. After confirming the absence of any residual starting material peak, the solvent was filtered under reduced pressure, and the resin was washed five times with 15 mL of N,N-dimethylformamide to obtain N-ε-AFB2a-N-α-Boc-lysine resin. The intermediate did not require purification and was directly fed into the next step.

[0086] (4) Synthesis of N-ε-AFB1-N-α-Boc-lysine resin:

[0087] Add 30 mL of N,N-dimethylformamide to a solid-phase synthesis tube of N-ε-AFB2a-N-α-Boc-lysine resin (1.125 mmol / g). Weigh peracetic acid (0.33 g, 4.26 mmol) and place it in a centrifuge tube. Add 3 mL of N,N-dimethylformamide and slowly add it dropwise to the solid-phase synthesis tube at 0°C. After the addition is complete, transfer the tube to a constant-temperature shaker and shake at 0°C and 170 rpm for 1 hour. After the reaction is complete, take a trace amount of resin and cleave it with 100 μL of a mixture of trifluoroacetic acid and dichloromethane (95:5 volume ratio). Monitor the cleavage with HPLC. If no raw material peak remains, filter the solvent under reduced pressure and wash five times with 15 mL of N,N-dimethylformamide to obtain N-ε-AFB1-N-α-Boc-lysine resin. The intermediate does not require purification and can be directly added to the next step.

[0088] (5) Synthesis of aflatoxin lysine adduct AFB1-Lys:

[0089] Using N-ε-AFB1-N-α-Boc-lysine resin (1.125 mmol / g) as the starting material, 20 mL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio of trifluoroacetic acid to dichloromethane 95:5) was added to a solid-phase synthesis tube as the lysis buffer. This step uses a high-concentration lysis buffer to remove the N-α-Boc protecting group while cleaving the resin. The mixture was transferred to a constant-temperature shaker and shaken at 25°C and 170 rpm for 30 minutes. After the reaction, the lysis buffer was monitored by HPLC. If no residual starting material peak was observed, the filtrate was collected by vacuum filtration. The filtrate was washed twice with 10 mL of dichloromethane and collected. Most of the solvent was removed by rotary evaporation at 0.08-0.09 MPa vacuum, 40 rpm, and 25°C. The filtrate was then evaporated until 1 / 4 of the original solvent remained. This residue was then added dropwise to 50 mL of ice-cold diethyl ether and cooled at -20°C. A large amount of white precipitate was observed in the reaction solution. The precipitate was transferred to a 50 mL centrifuge tube and centrifuged at 5 °C, 10,000 rpm for 5 min. The supernatant was discarded, and the filter cake was washed 2-3 times with ice-cold ether. The resulting solid was the crude peptide product. The sample to be purified was dissolved in acetonitrile / water (1:1, v / v) at a concentration of 15 mg / mL, ensuring complete dissolution. The sample solution was filtered through a 0.22 μm organic phase filter to remove particulate impurities and prevent column clogging. A C18 reversed-phase column (5 μm, 30 × 150 mm) was used. Mobile phase A was ultrapure water (containing 0.1% trifluoroacetic acid by volume), and mobile phase B was preparative grade acetonitrile (containing 0.1% trifluoroacetic acid by volume). The mobile phases were degassed by sonication for 15 min and then filtered through a 0.22 μm filter. The preparative HPLC system was set to a flow rate of 25 mL / min, with an initial mobile phase composition of 5% mobile phase B. The elution time was set to 40 min, with a gradient of 5%–30% mobile phase B (0–25 min), 30%–50% mobile phase B (25–35 min), and 50%–5% mobile phase B (35–40 min). The product was collected using a fraction collector, lyophilized, and its HPLC purity was determined to be 99%, yielding a high-purity aflatoxin lysine adduct AFB1-Lys. The product was a pale yellow solid, 150 mg in size, with an overall yield of 15%.

[0090] Example 3 The preparation methods (1) and (2) of the aflatoxin lysine adduct based on solid-phase synthesis provided in this embodiment are the same as those in Example 1. The synthesis of the aflatoxin lysine adduct AFG1-Lys in step (3) differs from step (3) of the synthesis method in Example 1 in that the aflatoxin used in the reaction of this embodiment is AFG2a and the amount of experimental feed is reduced by 10 times.

[0091] This embodiment provides a method for preparing aflatoxin lysine adduct based on solid-phase synthesis, including the following steps: (3) Synthesis of N-ε-AFG2a-N-α-Boc-lysine resin:

[0092] In solid-phase synthesis, the molar ratio of feed in subsequent steps is calculated based on the degree of substitution of the resin in step (1) (i.e., the number of moles of functional groups on a unit mass of resin).

[0093] Using N-α-Boc lysine resin (1.245 mmol / g) as the starting material, AFG2a standard (75 mg, 0.213 mmol) was weighed and placed in a centrifuge tube. 10 mL of N,N-dimethylformamide and N,N-diisopropylethylamine (54.9 mg, 0.426 mmol) were added, and the mixture was shaken to form a clear homogeneous solution. This solution was then added to a solid-phase synthesis tube and transferred to a constant-temperature shaker at 25°C and 170 rpm for 4 hours. After the reaction, a trace amount of resin was cleaved using 100 μL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio 5:95) as lysis buffer, and the cleavage was monitored by HPLC. After confirming the absence of any residual starting material peaks, the solvent was filtered under reduced pressure, and the resin was washed five times with 5 mL of N,N-dimethylformamide to obtain N-ε-AFG2a-N-α-Boc-lysine resin. The intermediate did not require purification and was directly fed into the next step.

[0094] (4) Synthesis of N-ε-AFG1-N-α-Boc-lysine resin:

[0095] Add 10 mL of N,N-dimethylformamide to a solid-phase synthesis tube of N-ε-AFG2a-N-α-Boc-lysine resin (1.245 mmol / g). Weigh the oxidant m-chloroperoxybenzoic acid (74 mg, 0.426 mmol) into a centrifuge tube, add 1 mL of N,N-dimethylformamide, and slowly add it dropwise to the solid-phase synthesis tube at 0°C. After the addition is complete, transfer the tube to a constant-temperature shaker and shake at 170 rpm for 1 hour at 0°C. After the reaction is complete, take a trace amount of resin and cleave it with 100 μL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio of trifluoroacetic acid to dichloromethane 5:95). Monitor the reaction by HPLC. If no residual starting material peak is observed, filter the solvent under reduced pressure and wash five times with 5 mL of N,N-dimethylformamide to obtain N-ε-AFG1-N-α-Boc-lysine resin. The intermediate does not require purification and can be directly added to the next step.

[0096] (5) Synthesis of aflatoxin lysine adduct AFG1-Lys:

[0097] Using N-ε-AFG1-N-α-Boc-lysine resin (1.245 mmol / g) as the starting material, 10 mL of a mixture of trifluoroacetic acid and dichloromethane (volume ratio of trifluoroacetic acid to dichloromethane 3:7) was added to a solid-phase synthesis tube. A higher concentration of lysis buffer was used in this step to remove the N-α-Boc protecting group while cleaving the resin. The mixture was transferred to a constant-temperature shaker and shaken at 25°C and 170 rpm for 30 minutes. After the reaction, the lysis buffer was monitored by HPLC. If no residual starting material peak was observed, the filtrate was collected by vacuum filtration. The filtrate was washed five times with 5 mL of dichloromethane and collected. Most of the solvent was removed by rotary evaporation at 0.08–0.09 MPa vacuum, 40 rpm, and 25°C. The filtrate was evaporated until 1 / 4 of the original solvent remained. Then, 50 mL of ice-cold diethyl ether was added dropwise and cooled at -20°C. A large amount of white precipitate was observed in the reaction solution. The precipitate was transferred to a 50 mL centrifuge tube and centrifuged at 5 °C, 10,000 rpm for 5 min. The supernatant was discarded, and the filter cake was washed 2-3 times with ice-cold ether. The resulting solid was the crude peptide product. The sample to be purified was dissolved in acetonitrile / water (1:1, v / v) at a concentration of 15 mg / mL, ensuring complete dissolution. The sample solution was filtered through a 0.22 μm organic phase filter to remove particulate impurities and prevent column clogging. A C18 reversed-phase column (5 μm, 30 × 150 mm) was selected. Mobile phase A was ultrapure water (containing 0.1% trifluoroacetic acid by volume), and mobile phase B was preparative grade acetonitrile (containing 0.1% trifluoroacetic acid). The mobile phases were degassed by sonication for 15 min and filtered through a 0.22 μm filter. The preparative HPLC system was set to a flow rate of 25 mL / min, with an initial mobile phase composition of 5% mobile phase B. The elution time was set to 40 min, with a gradient of 5%–30% mobile phase B (0–25 min), 30%–50% mobile phase B (25–35 min), and 50%–5% mobile phase B (35–40 min). The product was collected using a fraction collector, lyophilized, and its HPLC purity was determined to be 99%, yielding high-purity aflatoxin lysine adduct AFG1-Lys. The product was a pale yellow solid, 56 mg in size, with an overall yield of 57%.

[0098] Figure 1 The image shows the ¹H NMR spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of this invention. Figure 1 It can be seen that the compound has a single configuration without isomers (AFB1-ε-LYS).

[0099] Figure 2The liquid phase spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of this invention is shown below. From top to bottom, peak 1 represents the aflatoxin B1-lysine peak eluted by liquid phase gradient, peak 2 represents the hydrogenation peak of aflatoxin B1-lysine with a molecular weight of 456.72-457.72, and peak 3 represents the sodium addition peak of aflatoxin B1-lysine with a molecular weight of 478.74-479.74. The elution time of all three peaks is 2.84 min, which verifies that they are the correct liquid phase peaks of aflatoxin B1-lysine.

[0100] Figure 3 This is the mass spectrum of aflatoxin B1-lysine prepared in step (5) of Example 1 of the present invention. The molecular weight 456.72-457.72 is the hydrogenation peak of aflatoxin B1-lysine, and the molecular weight 478.74-479.74 is the sodium addition peak of aflatoxin B1-lysine.

[0101] Figure 4 The liquid phase spectrum of aflatoxin G1-lysine prepared in step (5) of Example 3 of the present invention represents the peak of aflatoxin G1-lysine obtained by liquid phase gradient elution.

[0102] Figure 5 This is the mass spectrum of aflatoxin G1-lysine prepared in step (5) of Example 3 of the present invention. The molecular weights 472.86-473.37 represent the hydrogenation peak of aflatoxin G1-lysine, and 495.26-496.27 represent the sodium addition peak. This confirms the correct mass spectrometric molecular weight of aflatoxin G1-lysine.

[0103] Figure 6 The image shows the ¹H NMR spectrum of the control isomer aflatoxin B1-lysine. It is the spectrum of AFB1-α-LYS.

Claims

1. A process for the preparation of a solid phase synthesis based aflatoxin lysine adduct characterized in that, Includes the following steps: (1) Synthesis of amino acid carrier resin: The carrier resin was swollen with a good solvent to obtain swollen resin; N-α-Boc-N-ε-Fmoc-lysine was weighed and added to an organic solvent and an organic base. After shaking to form a clear homogeneous solution, it was added to the swollen resin for reaction. The mixture was shaken, filtered, washed, and sealed with a blocking solution. The mixture was shaken, filtered, and washed to obtain N-α-Boc-N-ε-Fmoc-lysine resin. (2) Deprotection of amino acid resin: Using the N-α-Boc-N-ε-Fmoc-lysine resin prepared in step (1) as raw material, add piperidine organic solvent solution to treat 1 to 3 times to remove Fmoc protecting group, wash, and obtain N-α-Boc-lysine resin. (3) Cycloning: Weigh aflatoxin dialdehyde, add it to an organic solvent and an organic base, shake to form a clear homogeneous solution, and then add it to the N-α-Boc-lysine resin prepared in step (2). Shake, filter, and wash to obtain N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin. (4) Oxidation: Add organic solvent and oxidant to the N-ε-aflatoxin dialdehyde-N-α-Boc-lysine resin prepared in step (3), shake, filter, and wash to obtain N-ε-aflatoxin-N-α-Boc-lysine resin. (5) Cutting and purification: Using the N-ε-aflatoxin-N-α-Boc-lysine resin prepared in step (4) as raw material, acid lysis buffer is added to cut the aflatoxin lysine adduct on the N-ε-aflatoxin-N-α-Boc-lysine resin and remove the Boc protecting group at the same time. After filtration, vacuum rotary evaporation is performed, and the filtrate is added to the precipitant. After low-temperature centrifugation, the supernatant is discarded and the crude product is washed to obtain the crude product. The crude product is purified to obtain the aflatoxin lysine adduct.

2. The process for the preparation of aflatoxin lysine adduct according to claim 1, characterized in that, In step (1), the sample-carrying resin is selected from either CTC resin or Wang resin.

3. The process for the preparation of aflatoxin lysine adduct according to claim 1, characterized in that, The organic solvents in steps (1), (2), (3) and (4) are each independently selected from any one or a mixture of several of dichloromethane, dichloroethane, trichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone.

4. The process for preparing aflatoxin lysine adduct according to claim 1, characterized in that, In steps (1) and (3), each organic base is independently selected from any one or a mixture of several of N,N-diisopropylethylamine, triethylamine, triethanolamine, and pyridine.

5. The method for preparing aflatoxin-lysine adduct according to claim 1, characterized in that, In step (5), the precipitant is icy ethyl ether or icy methyl tert-butyl ether.

6. The process for preparing aflatoxin lysine adduct according to claim 1, characterized in that, In step (5), the purification conditions are as follows: using a C18 reversed-phase column as the stationary phase, and using an aqueous solution containing 0.05% to 0.15% trifluoroacetic acid and an acetonitrile solution containing 0.05% to 0.15% trifluoroacetic acid as the mobile phase for gradient elution.

7. Aflatoxin lysine adduct prepared according to the process of any one of claims 1 to 6, characterized in that, The adduct is AFB1-Lys or AFG1-Lys, and its HPLC purity is not less than 95%.

8. A standard of aflatoxin lysine adduct, characterized in that, The standard contains an aflatoxin lysine adduct prepared by any one of claims 1-6 or an aflatoxin lysine adduct as described in claim 7.

9. The use of the aflatoxin lysine adduct prepared by any one of claims 1-6 or the aflatoxin lysine adduct as described in claim 7 in the preparation of standards for the detection of aflatoxin exposure biomarkers.

10. The use of the aflatoxin lysine adduct prepared by any one of claims 1-6 or the aflatoxin lysine adduct as described in claim 7 in the preparation of reagents or kits for food safety testing or clinical medical diagnosis.

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